The present invention relates to settable compositions for general purpose concrete construction containing Class-F fly ash, Class-C fly ash or slag, and cement kiln dust (CKD) as a substantial replacement for Portland cement conventionally used in such compositions. The compositions of the present invention provide high early strength thereby allowing the concrete structure to be put into service sooner, reducing labor cost, and allowing precast concrete and concrete masonry manufacturers to achieve rapid form and mold turnover.

This invention relates to the field of settable compositions for general purpose concrete construction containing Class-F fly ash, Class-C fly ash or slag, and cement kiln dust (CKD) as a substantial replacement for Portland cement conventionally used in such compositions.
This invention is concerned with the utilization of four industrial by-products; namely, Class-F fly ash, Class-C fly ash, blast furnace slag, and cement kiln dust (CKD) in general purpose concrete-making composition. When finely divided or pulverized coal is combusted at high temperatures, for example, in boilers for the steam generation of electricity, the ash consisting of the incombustible residue plus a small amount of residual combustible matter, is made up of two fractions, a bottom ash recovered from the furnace or boiler in the form of a slag-like material and a fly ash which remains suspended in the flue gases from the combustion until separated therefrom by known separation techniques, such as electrostatic precipitation. This fly ash is an extremely finely divided material generally in the form of spherical bead-like particles, with at least 70% by weight passing a 200 mesh sieve and has a generally glassy state, resulting from fusion or sintering during combustion. As recognized in the American Society of Testing Materials (ASTM) specification designations C618-00 entitled xe2x80x9cFly Ash and Raw or Calcined Natural Pozzolan for Use as a Mineral Admixture in Portland Cement Concretexe2x80x9d and D5370-96 entitled xe2x80x9cStandard Specification for Pozzolanic Blended Materials in Construction Application,xe2x80x9d fly ash is subdivided into two distinct classifications; namely, Class-F and Class-C. The definitions of these two classes are as follows:
xe2x80x9cClass-Fxe2x80x94Fly ash normally produced from burning anthracite or bituminous coal that meets the applicable requirements for this class as given herein. This class fly ash has Pozzolanic properties.
Class-Cxe2x80x94Fly ash normally produced from lignite or subbituminous coal that meets the applicable requirements for this class as given herein. This class of fly ash, in addition to having pozzolanic properties, also has some cementitious properties. Some Class-C fly ashes may contain lime contents higher than 10%.xe2x80x9d
The latter reference to xe2x80x9cpozzolanic propertiesxe2x80x9d refers to the capability of certain mixtures that are not in themselves cementitious, but are capable of undergoing a cementitious reaction when mixed with calcium hydroxide in the presence of water. Class-C fly ash possesses direct cementitious properties as well as pozzolanic properties. ASTM C618-00 is also applicable to natural pozzolanic materials that are separately classified as Class N but are not pertinent here.
As the above quotation indicates, the type of coal to be combusted generally determines which class fly ash results, and the type of coal in turn is often dependent on its geographic origin. Thus, Class-C fly ash frequently results from coals mined in the Midwest; whereas Class-F fly ash often comes from coals mined in the Appalachian region. The ASTM specification imposes certain chemical and physical requirements upon the respective fly ash classifications which are set forth in U.S. Pat. No. 5,520,730 which is incorporated herein by reference.
CKD, on the other hand, is a by-product of the production of Portland cement clinkers by the high temperature furmacing of appropriate raw materials, typically mixtures of limestone and clay or a low grade limestone already containing a sufficient quantity of argillaceous materials often with added quantities of lime to adjust the final composition. The resultant clinkers are pulverized by grinding with gypsum to a high degree of fineness and these particles upon admixture with sand gravel and sufficient water undergo a cementitious reaction and produce the solid product generally referred to as concrete, which exhibits high compressive strength and is thus highly useful in construction of a great variety of building or supporting structures. Generally, rotary furnaces are used for producing Portland cement clinkers and a certain quantity of finely divided dust is produced as a by-product that is carried off in the flue gases from such furnaces. The dust content can range from about 5% of the clinkers output in so-called wet process plants up to as high as 15% in dry process plants. The suspended dust is removed by various separating techniques and remains as a by-product of the cement making operation. Part of the CKD can be returned to the furnace as recycled raw material, but it is not readily reincorporated into clinker formation and, in addition, tends to excessively elevate the alkalinity of the ultimate Portland cement.
Blast furnace slag is a by-product from the production of iron in a blast furnace; silicon, calcium, aluminum, magnesium and oxygen are the major elemental components of the slag. Blast furnace slags include air-cooled slag resulting from solidification of molten blast furnace slag under atmospheric conditions; granulated blast furnace slag, a glassy granular material formed when molten blast furnace slag is rapidly chilled as by immersion in water; and pelletized blast furnace slag produced by passing molten slag over a vibrating feed plate where it is expanded and cooled by water sprays, whence it passes onto a rotating drum from which it is dispatched into the air where it rapidly solidifies to spherical pellets. In general the glass content of the slag determines the cementitious character, rapidly cooled slags have a higher glass content and are cementitious; slowly cooled slags are non-glassy and crystalline and thus do not have significant cementitious properties.
The quantities of these by-product materials that are produced annually are enormous and are likely only to increase in the future. As petroleum oil as the fuel for the generation of electricity is reduced because of conservation efforts and unfavorable economics, and as political considerations increasingly preclude the construction of new nuclear power electrical generating facilities, or even the operation of already completed units of this type, greater reliance will necessarily fall on coal as the fuel for generating electricity. As of 1979, the amount of CKD was estimated as accumulating at a rate of 4-12 million tons per year in the United States alone, whereas the amount of Class-F fly ash that is available is estimated to be about five times what can be readily utilized. The estimated yearly total production of coal ash in the U.S. is about 66.8 million tons, while the yearly total coal ash sales in the U.S. is about 14.5 million tons. Further, in Canada, the recovery of copper, nickel, lead and zinc from their ores produces over twelve million tons of slag per year, which usually accumulated near the smelters without significant use. Obviously, there is an urgent growing need to find effective ways of employing these unavoidable industrial by-products since otherwise they will collect at a staggering rate and create crucial concerns over their adverse environmental effect.
Various proposals have already been made for utilizing both fly ash and CKD. According to Lea (1971), The Chemistry of Cement and Concrete, Chemical Publishing Company, Inc., page 421 et seq., fly ash, i.e., Class-F type, from boilers was first reported to be potentially useful as a partial replacement for Portland cement in concrete construction about 50 years ago, and its utilization for that purpose has since become increasingly widespread. It is generally accepted that the proportion of Portland cement replaced by the usual fly ash should not exceed about 20% to avoid significant reduction in the compressive strength of the resultant concrete, although some more cautious jurisdictions may impose lower limits, e.g., the 15% maximum authorized by the Virginia Department of Highways and Transportation (VDHT). As described in Lea on page 437, the substitution of fly ash tends to retard the early rate of hardening of the concrete so that the concrete shows up to a 30% lower strength after seven days testing and up to a 25% lower strength after 28 days of testing, but in time the strength levels equalize at replacement levels up to 20%. Increasing the substitution quantity up to 30% gives more drastic reduction in the early compression values plus an ultimate reduction of at least about 15% after one year.
The limited substitution of fly ash for Portland cement in concrete formulations has other effects beyond compressive strength changes, both positive and negative. The fly ash tends to increase the workability of the cement mix and is recognized as desirably reducing the reactivity of the Portland cement with so-called reactive aggregates. On the other hand, fly ash contains a minor content of uncombusted carbon that acts to absorb air entrained in the concrete. Because entrained air increases the resistance of the hardened concrete to freezing, such reduction is undesirable but can be compensated for by the inclusion as an additive of so-called air-entraining agents.
Dodson, et al. in U.S. Pat. No. 4,210,457, while recognizing the accepted limit of 20% replacement with fly ash of the Portland cement in concrete mixes, proposed the substitution of larger amounts, preferably 50% or more, of the Portland cement with particular selected fly ashes having a combined content of silica, alumina and ferric oxide content, less than 80% by weight, and a calcium oxide content exceeding 10%, based on five samples of such ashes, varying from about 58-72% combined with a calcium oxide range of about 18-30%. Six other fly ash samples that are not suitable at the high replacement levels of 50% or more were shown to vary in the combined oxide content from about 87-92% and in calcium oxide content from about 4 to about 8%. Evaluating these values against the ASTM C618-00, one observes that the acceptable fly ashes came under the Class-C specifications, while the unacceptable ashes fell in the Class-F specification. Thus, this patent in effect establishes that Class-C fly ashes are suitable for substantially higher levels of replacement for Portland cement in concrete mixes than are Class-F fly ashes, and this capacity is now generally recognized, with Class-C fly ashes being generally permitted up to about a 50% replacement level while maintaining the desirable physical properties of the concrete especially compressive strength.
In U.S. Pat. No. 4,240,952, Hulbert, et al. while also acknowledging the generally recognized a permissible limit of Class-F fly ash replacement for Portland cement of 20%, proposed replacement of at lest 50% up to 80%, provided the mix contained as special additives about 2% of gypsum and about 3% of calcium chloride by weight of the fly ash. The fly ash described for this purpose, however, was a Class-C fly ash analyzing about 28% calcium oxide and combined silica, alumina and ferric oxide content of about 63%. With up to 80% of this fly ash and the specified additives, compressive strengths comparable to straight Portland cement were said to be generally achievable. In one example using 140 pounds Portland cement and 560 pounds of fly ash (20:80 ratio) with conventional amounts of coarse and fine aggregate, and water and including the requisite additives, compressive strengths tested at 3180 psi for 7 days, 4200 psi for 14 days and about 5000 psi at 28 days.
In U.S. Pat. No. 4,018,617 and U.S. Pat. No. 4,101,332, Nicholson proposed the use of mixtures of fly ash (apparently Class-F in type), cement kiln dust and aggregate for creating a stabilized base supporting surface replacing conventional gravel or asphalt aggregate stabilized bases in road construction wherein the useful ranges were fly ash 6-24%, CKD 4-16% and aggregate 60-90%, with 8% CKD, 12% fly ash and 80% aggregate preferred. Compressive strength values for such measures as revealed in the examples varied rather erratically and generally exhibited only small increases in compression strength over the 7 to 28 day test period. Among the better results were for the preferred mixture wherein the values increased from about 1100 psi at 7 days to 1400 psi at 28 days. The addition of a small amount of calcium chloride improved those values by about 200 psi. On the other hand, the addition of 3% of lime stack dust recovered from a lime kiln significantly reduced the results to about 700 psi at 7 days to 900-1300 psi at 28 days. Elimination of the aggregate reduced the strength to a fraction of the values otherwise obtained, a mixture of 12% CKD and 88% fly ash alone showing strength values of only about 190-260 psi over the 28-day test period. Similarly, the choice of a finely divided aggregate such as fill sand resulted in about the same fractional level of strength values in the range of about 140-230 psi. A combination of finely divided and coarse aggregate in approximately equal amounts reduced the compressive strength values by about xc2xd with virtually no change over the test period, giving values ranging from 650-750 psi, except where 1% of Type 1 Portland cement was included which restored the strength values to about their original level, except at the initial 7 days period where the strength values were about 800-900 psi, increasing at 28 days to about 1200-1600 psi. Curiously, the best strength results were attained when 11.6% fly ash was combined with 3.4% lime with the balance crushed aggregate, the CKD being omitted entirely, for which the strength values while starting at a lower level of about 850-950 at 7 days increased to about 1700 psi at 28 days.
The combination of fly ash and lime stack dust incidentally mentioned in the later patent was explored further by Nicholson in U.S. Pat. No. 4,038,095 which concerns mixtures of about 10-14% fly ash, about 5-15% lime stack dust with the balance aggregate in the range of 71-85%. Somewhat inexplicably, the compressive results reported here for such mixtures do not reach the high level specified in the first two patents, the strength values specified being only about 1000 psi with the more general levels well below that depending on particular proportions.
In U.S. Pat. No. 4,268,316, Wills, Jr., discloses the use of mixtures of kiln dust and fly ash as a replacement for ground limestone and gypsum for forming a mortar or masonry cement, using proportions of about 25-55% Portland cement, about 25-65% CKD and 10-25% fly ash. When these mortar formulations were mixed with damp sand in the proportions of about one part cement mixture to 2.5-3 parts sand, compression strengths comparable to those of standard masonry cement composed of 55% cement clinkers 40% limestone and 5% gypsum were shown for mixtures containing 50% cement, 24-40% CKD and 15-25% fly ash. Inexplicably, in one example, when the cement content was increased to 55% with 35% CKD and 10-% fly ash, the compressive strengths dropped by about 30-40% at both the 7 day and 28 day ages to levels inferior to the standard material. As the cement content was decreased, with corresponding increases in the CKD, the compressive strength values dropped drastically. On the other hand, in another similar example mixtures containing 55% cement, 35% CKD and 10% fly ash proved superior, particularly at the 28 day age, in compressive strength, to mixtures containing 50% cement, 35% fly ash and 15% CKD as well as other standard masonry cements containing 50% cement, 47% limestone and 3% gypsum. Indeed, strength values dropped about 40% for the mixtures having a 5% reduction in cement and a corresponding 5% increase in the fly ash to values definitely inferior to the standard cements. Similar variations were shown under laboratory test conditions for comparable 50/35/15 mixtures dependent on the source of the fly ash while under actual construction conditions for the same mixtures, compressive strength values were reduced by about 50% for both the conventional masonry cement containing 55% Portland cement and comparable mixtures within the patented concept. The fly ash here was preferably Class-F with Class-C materials being less desirable.
In U.S. Pat. No. 4,407,677, Wills, Jr., went on to teach that in the manufacture of concrete products such as blocks or bricks, the fly ash usually employed in combination with Portland cement therein could be replaced in its entirety by CKD with modest improvement in early compressive strength values for such products. Thus, at one day and two day tests compressive strength values were shown of about 500-800 psi, but were said to increase to about 1200 psi after 28 days. The mixes disclosed here contained 0.4-0.9 parts cement, about 0.1-0.6 parts CKD and 10-12 parts aggregate combining both fine and coarse materials, such as expanded shale and natural sand in a weight ratio of 80/20. Masonry cements generally develop at least about 95% of their strength properties at 28 days age so that additional aging of the patented products would not be expected to result in any significant increase in their compressive strength values.
In U.S. Pat. No. 5,106,422, Bennett et al. teaches a self-hardening backfill material that utilizes Class-C fly ash as a primary constituent in conjunction with other fly ashes, such as Class-F fly ash, or other filler materials. The material can attain compressive strength of about 20 psi within about four hours, which is about 25 to 40 percent of its 28-day strength. The material, however, does not use cement and thus has low strength and is not useful in applications requiring concrete.
None of the above patents addresses the issue of early strength of concrete; therefore, there remains a need for concrete mixes containing fly ash with high early strength, because the addition of fly ash to concrete often results in slow setting. There are many advantages for having high early strength, such as allowing the concrete structure to be put into service sooner, thereby reducing labor cost, and allowing precast concrete and concrete masonry manufacturers to achieve rapid form and mold turnover.
An object of the instant invention relates a settable composition for improved early strength comprising cement, Class-F fly ash, Class-C fly ash, and CKD. In a preferred embodiment, the cement is present in an amount greater than about 50% by weight, the Class-F fly ash is present in an amount of about 1 percent to about 10 percent by weight, the Class-C fly ash is present in an amount of about 5 percent to about 25 percent by weight, and CKD is present in an amount of about 1 percent to about 15 percent by weight.
A further object of the instant invention relates a settable composition for improved early strength comprising cement, Class-F fly ash, slag, and CKD. In a preferred embodiment, the cement is present in an amount greater than about 50% by weight, the Class-F fly ash is present in an amount of about 2 percent to about 11 percent by weight, the slag is present in an amount of about 1 percent to about 15 percent by weight, and CKD is present in an amount of about 3 percent to about 20 percent by weight.
Methods of making concrete from the above compositions are also disclosed.
Several different types of Portland cement are available and all are useful with the present invention. Type I is the general purpose variety and is most commonly employed but Type III is preferable for early strength application. Commercial blended cements, such as Type I-P, wherein 20% Class-F fly ash is blended with 80% by weight Portland cement clinker during pulverization should be avoided.
Any standard or common Class-F fly ash obtained from boilers and like furnaces used for the combustion of pulverized coal, particularly of a bituminous or anthracite type, and especially from coal-fired, steam-generating plants of electrical utilities, is suitable for use as the Class-F fly ash component of this invention. Such fly ash should have a combined silica, alumina and ferric oxide content of at least about 70% and preferably 80% or higher by weight and a lime (CaO) content below about 10%, usually about 6% by weight or less.
Any standard or common Class-C fly ash obtained from the burning of lignite or subbituminous coal is suitable for use as the Class-C fly ash component of this invention. Such Class-C fly ash generally contains more calcium and less iron than Class-F fly ash and has a lime content in the range of 15% to 30%.
Similarly, any common cement kiln dust (CKD) that is produced as a by-product during the industrial production of Portland cement would in principle be suitable for purposes of this invention. One specific CKD, obtained as a matter of convenience, from the Tarma Lone Star Cement Company cement plant at Roanoke, Va., has previously been found useful in the cement composition. Other various CKD can be found in the patent literature. For example, U.S. Pat. No. 4,018,617 to Nicholson mentioned previously, analyzed nine different samples of CKD.
Likewise, any blast furnace slag is appropriate for the present invention. Slag is a non-metallic coproduct produced in the production of iron in a blast furnace. It consists primarily of silicates, aluminosilicates and calcium-alumina-silicates. The molten slag usually comprises about twenty percent by mass of iron production. Different forms of slag products are produced depending on the method used to cool the molten slag. These products include air-cooled blast furnace slag, expanded or foamed slag, palletized slag, and granulated blast furnace slag. Granulated blast furnace slag satisfying ASTM 989 specification is preferred.
As will be established later, within the above limits for the compositions of the invention, the concrete produced therefrom exhibit substantially comparable or superior properties for use in general purpose cement construction, especially one-day compressive strength to corresponding all Portland cement mixes. This being the case, economic considerations may be an important factor in selecting a specific mix within such ranges. Under present market conditions, and dependent upon transportation distances from the available sources of the two components, CKD can be purchased somewhat more cheaply than can a standard Class-F fly ash. For example, fly ash might be purchased at a cost of $20.00 per ton including transportation expense of about $7.00 per ton; whereas CKD can be purchased for about $9.00 per ton including about $4.00 transportation expense. Where the relative expense significantly favors one of the products, such as CKD, it is economically advantageous to utilize a larger amount of the cheaper constituent. Thus a mix having CKD and Class-F fly ash would be cheaper to produce than a mix having only Class-F fly ash.
Concrete mixes using the present invention may also contain aggregate materials. The choice of aggregate material for concrete mixes using the present blends will pose not problem to the person skilled in the design of such mixes. The coarse aggregate should have a minimum size of about xe2x85x9c inch and can vary in size from that minimum up to one inch or larger, preferably in gradations between these limits. Crushed limestone, gravel and the like are desirable coarse aggregates, and the material selected in any case should exhibit a considerable hardness and durability inasmuch as crumbly, friable aggregates tend to significantly reduce the strength of the ultimate concrete. The finely divided aggregate is smaller than xe2x85x9c inch in size and again is preferably graduated in much finer sizes down to 200-sieve size or so. Ground limestone, sand and the like are common useful fine aggregates.
In accordance with the present invention, silica fume can also be added to the cement mixture to achieve high strength and chloride protection for the concrete. Silica fume is preferably used from 3-12 percent of the amount of cement that is being use in the mixture.
Other additives can also be used in accordance with the present invention, including, but is not limited to, water reducers, accelerators, air entrainment agents, as well as other additives that is commonly used in the concrete industry.
The mixes of the invention are prepared by homogeneously and uniformly mixing all of the mix ingredients including the Class-F fly ash, Class-C fly ash, slag, and CKD. The Class-F fly ash has a specific gravity of about 2.25, while that of CKD is around 2.70. These relatively small differences in specific gravities do not create any unusual problems in the preparation of the present compositions and any of the usual mixing techniques commonly employed in the concrete mix industry are suitable. The ultimate compositions are no more susceptible to undergo separation during handling and storage than are ordinarily concrete mixes. They can be transported and stored in the same manner as the ordinary mixes, as can the individual ingredients. The storage containers should, of course, be closed to protect the contents thereof from weather.
The following examples are given to illustrate the present invention. It should be understood that the invention is not limited to the specific conditions or details described in these examples.
The results in the following examples were actually obtained by preliminarily blending, in each case, the Class-F fly ash, Class-C fly ash, slag, and CKD together in accordance with the concept of the prior application and combining the blend with the other mix ingredients. However, the results would be identical if the same proportionate amount for each of the component was added separately to the remaining mix ingredients and the proportionate amounts of the Class-F fly ash, Class-C fly ash, slag, and CKD have been expressed in each case in terms of their relative weight percentages of the particular mix.

Settable composition containing cement kiln dust


In one embodiment, a method includes receiving a request for evaluating driving data included in a data log, accessing, based on the request, an evaluation configuration file that includes a metric-calculation configuration specifying one or more metric calculators configured to generate one or more output metrics from the driving data and a validation configuration configured to validate the one or more output metrics, instantiating the one or more metric calculators specified by the metric-calculation configuration included in the evaluation configuration file, determining particular driving data from the driving data included in the data log based on the one or more instantiated metric calculators, and generating the one or more output metrics for the particular driving data by using the instantiated one or more metric calculators.

Evaluation is a systematic determination of a subject's merit, worth and significance, using criteria governed by a set of standards. It can assist an organization, program, design, project or any other intervention or initiative to assess any aim, realizable concept/proposal, or any alternative, to help in decision-making; or to ascertain the degree of achievement or value in regard to the aim and objectives and results of any such action that has been completed. The primary purpose of evaluation, in addition to gaining insight into prior or existing initiatives, is to enable reflection and assist in the identification of future change. Evaluation is often used to characterize and appraise subjects of interest in a wide range of human enterprises, including the arts, criminal justice, foundations, non-profit organizations, government, health care, and other human services. It is long term and done at the end of a period of time.

Evaluating driving data with a modular and configurable evaluation framework


A system for balancing network traffic among virtual machines includes a first virtual machine executed by a first physical server connected to a physical network and a second virtual machine executed by a second physical server connected to the physical network. A gateway device is configured to route network traffic through the physical network to and from the first and second virtual machines. A load balancer module is executed by the gateway device. The load balancer module is configured to: at least partially decode the network traffic to identify a destination address of the network traffic; determine if the destination address is the address of a load-balanced virtual machine; and if the destination address is the address of a load-balanced virtual machine, route the network traffic to a destination virtual machine according to a load-balancing scheme.

As is known in the art, a data center is a facility used to house computer systems and associated components, such as telecommunications and storage systems. It generally includes redundant or backup power supplies, redundant data communications connections, redundant storage devices, environmental controls (e.g., air conditioning, fire suppression) and security devices.


A spout is used for confining the grain flow from an auger. The auger may be a combine, grain cart unloading auger or a grain auger. The spout includes a chute that mounts on the auger tube. An overflow opening in the chute allows grain or other matter to flow out of the spout if the outlet of the chute is plugged. This prevents filling and overloading of the auger and consequent auger damage.

The present invention relates to auger discharge control and more particularly to a flow control spout for an auger, especially the unloading auger of a combine harvester.
Grain or seed is delivered from a combine harvester to the box of a grain truck by a delivery auger on the combine. The auger has a discharge at the end of the auger tube through which the grain is delivered. Because the grain is delivered at a high flow rate, there is a considerable spreading of the grain or seed as it leaves the auger discharge. This in turn leads to xe2x80x9csplashxe2x80x9d, spillage and wind loss. This may be controlled by a spout according to international patent application PCT/CA 98/00760. In use the spout is attached to the end of the auger to provide containment of the grain flow. It includes a chute having a receiving opening larger than the auger tube discharge opening and an outlet opening smaller than the receiver opening. A convergent peripheral wall extends from the inlet opening towards the outlet opening. When mounted on the auger tube, the receiving opening confronts the auger tube discharge opening and the peripheral wall slopes outwardly and downwardly from the end of the auger tube and under the discharge opening.
The sloping peripheral wall intercepts flow from the auger discharge, and redirects the flow to a direction with an outward component. This concentrates the flow from the auger without causing it to back up. In most cases, frusto-conical extensions are mounted on the chute as described in the application referenced above.
If, through inadvertence of an operator or any other cause, grain or seed is allowed to rise to the level of the spout discharge, the spout discharge may become closed, allowing the grain or seed to build up in the spout and then the auger. This will overload the auger and cause damage to the auger and its drive.
Another potential problem may arise if the spout comes into contact with an external object. For example, if the spout is positioned too low with respect to a truck being loaded and the truck hits the spout, damage may be caused to the spout, the auger or the truck.
The present invention is concerned with improvements in the spout that address these concerns.
According to one aspect of the present invention there is provided a spout for an auger having an auger tube with an outer end and an auger tube discharge opening adjacent the outer end, the spout comprising:
a chute having:
a receiving opening;
an outlet opening; and
a convergent peripheral wall extending from the inlet opening towards the outlet opening;
an overflow opening in the peripheral wall; and
a chute mount for mounting the chute on the auger tube with the receiving opening confronting the auger tube.
If the level of the material being conveyed rises to an undesirable level inside the chute for any reason, the excess will flow out of the overflow, preventing or delaying filling of the auger and giving the operator the opportunity to situation, either by removing a blockage or shutting down the auger.
The overflow is positioned out of the normal flow path of the material being conveyed, so that material is not lost through the overflow. It may be an outer overflow in an outwardly sloping outer side of the peripheral wall or an inner overflow in the inner side of a discharge collar forming the lowermost part of the peripheral wall, around the outlet opening. In preferred embodiments, both overflows are used.
The discharge collar is preferably frusto-conical. In use, the inner side of the chute intercepts the flow out of the auger discharge and directs it outwardly where it impinges upon and swirls about the inside of the collar to discharge downwardly from the spout.
To avoid the loss of material through the inner overflow, the peripheral wall may include a lip extending forwardly at the top of the discharge collar, over the inner overflow.
The inner overflow opening provides overflow capacity additional to that of the outer overflow and also an early visual warning to an operator of an overflow condition. This early warning can be augmented by placing a sensor in the inner overflow, for example a microswitch actuator at the bottom edge, to detect the onset of an overflow over the bottom edge.
The ingress of material into the chute through the outer overflow opening, high wind conditions and overshoot of material from the auger through the outer overflow can be concerns. These can be dealt with by providing a shield across the opening. This may be spaced sufficiently from the peripheral wall of the chute that there is no undue restriction of flow. Alternatively, the shield may be sufficiently light weight and freely movable that free flow is not impeded.
According to another aspect of the present invention there is provided a spout for an auger having an auger tube with an auger tube discharge opening on a bottom side of the auger tube, the spout comprising:
a chute having:
a receiving opening;
an outlet opening; and
a convergent peripheral wall extending from the inlet opening towards the outlet opening;
a chute mount for mounting the chute on the auger tube for rotation about the auger tube from a normal position in which the receiving opening confronts the auger tube discharge opening and the peripheral wall depends from the auger tube, and a released position in which the peripheral wall extends laterally from the auger tube; and
a chute centring device for biasing the spout to the normal position.
With this arrangement, engagement of the spout with an external object, for example a truck box, will rotate the spout to a sideways orientation to clear the obstruction with minimal, and preferably no, damage. The chute will automatically return to the normal position once it is released from the external engagement.
In preferred embodiments, the chute mount includes a substantially cylindrical hood for engagement over the outer end of the auger tube. The chute centring device preferably acts as a retainer holding the hood in place on the tube. The centring device may include a band clamped onto the auger tube and an end flange on the chute to engage the band and limit the travel of the chute along the auger tube. One or more resilient elements, for example springs or elastomeric straps, connect the band and the end flange to bias the chute to the normal position.


A method of sample displacement chromatography separation which comprises applying a multicomponent sample to one end of a chromatography bed comprising stationary phase material having affinity for components of the sample, causing components of the sample to become distributed along the chromatography bed by passage over the bed of a non-eluting mobile solvent phase, and recovering a desired component of the sample from at lest a portion of the chromatography bed, in which the sample components are applied to the chromatography bed in a non-homogeneous manner such that the concentration of at least one component with relatively low affinity for the stationary phase material is enhanced during an earlier part of sample application and/or the concentration of at least one component with relatively high affinity for the stationary phase material is enhanced during a later part of sample application.

This invention is concerned with chromatographic separation, more particularly with a new and improved method of sample displacement chromatography and applications thereof, as well as with apparatus useful in such methods.
During the last 10-15 years displacement chromatography has been suggested as an alternative to liquid chromatography using elution techniques. In elution chromatography, components of a sample are transported along a chromatography bed comprising stationary phase material, e.g. in a column, by a mobile solvent phase. The various components interact at different levels with the stationary phase material and are therefore separated into bands. Displacement chromatography, on the other hands, utilises as mobile phase a displacer solution which has higher affinity for the stationary phase material than do the sample components. In the case of column chromatography the sample components are thereby displaced and driven down the column ahead of the displacer front, competing for adsorption sites and separating into individual component bands as they proceed.
Whereas elution chromatography normally results in substantial dilution of the sample material, displacement chromatography permits recovery of sample components at significantly higher concentrations and generally makes more efficient use of the stationary phase material. However, displacement chromatography does suffer the disadvantage that, to achieve optimum results, operating conditions such as the composition, concentration and flow rate of the displacer solution must be tailored specifically to individual sample types.
Sample displacement chromatography is a self-displacement technique which was first proposed by Hodges et al. [J. Chromatogr. 444 (1988), pp. 349-362] for preparative purification of peptides by reversed phase HPLC, and which does away with the need for an extraneous displacer solution. The peptide components are applied to the column input and themselves compete for binding sites on the stationary phase as they are carried through the column or series of columns by an appropriate solvent. The more strongly binding components bind first and displace less strongly binding components to further along the column(s). The components are therefore separated according to their different degrees of hydrophobicity/hydrophilicity and thus their affinity for the stationary phase material. In a representative example a short pre-column is used to trap impurities which are more hydrophobic than the desired sample component, this latter being retained in and saturating the main column, while the more hydrophilic impurities are further displaced and so are washed out of the main column. It is suggested that the size of the pre-column may be adjusted to match the amount of hydrophobic impurities present in a particular sample, whilst the size of the main column may be adjusted to ensure maximum product retention and outflow of hydrophilic impurities.
Curiously Hodges et al. subsequently use gradient elution to recover the desired product from the main column, so that the advantageous potential of displacement chromatography for yielding relatively high concentration product solutions is lost. This would suggest that the initial component separation was incomplete.
Veeraragavan et al. [J. Chromatogr. 541 (1991), pp. 207-220] report application of the Hodges technique to purification of proteins using high performance anion exchange chromatography columns. The apparatus used was a low pressure fast protein liquid chromatographic system and again gradient elution was employed; this was presumably felt to be necessary in light of the observation that peak overlaps were a problem in the primary separation procedure. One and two column systems are specifically described, the former being applicable on what are said to be the rare occasions where the desired product is either the most or least strongly binding component. The possibility of using a multi-column system xe2x80x9cin which theoretically every component of the protein sample could be fixed to a column of the proper dimensionsxe2x80x9d is noted.
Multi-column HPLC systems for sample displacement chromatography have in fact been described by Hodges, inter alia in CA-A-2059114. A representative illustration shows the use of ten reversed phase HPLC columns or column segments connected in series for the purification of peptide samples; after the sample material has been loaded and distributed/separated over the train of columns, individual columns or segments may be eluted separately, without resort to gradient elution, the desired product component being recovered in substantially pure form from at least one such column or segment. Advantages of this process are said to be that (i) it allows ten-fold greater loading than comparable gradient elution separations; (ii) it involves minimal use of costly HPLC solvents; (iii) it requires minimal use of fraction analyses; (iv) it avoids the need to use displacer solutions during the actual separation; and (v) operating costs in terms of solvents, column packings and machine usage are much lower than typical gradient elutions.
The Hodges multi-column procedure does not appear to have been widely adopted and has been found in practice to give products with insufficient purity as a result of inadequate resolution of the product from closely related impurities. Moreover, by virtue of the need to operate HPLC procedures at high pressure, typically 80-200 bar, the apparatus required is necessarily complicated and expensive.
WO98/46623, the contents of which are incorporated herein by reference, describes sample displacement chromatography procedures which typically utilise low operating pressures and recover the desired product in a non-gradient manner. Such use of low operating pressures greatly simplifies apparatus requirements, permitting the use of simpler and less expensive pumps, taps, connectors and the like than are required for HPLC systems; the consequential low mobile phase flow rates also give rise to good separation of the sample components. By avoiding use of gradient elution the separation procedure also reduces solvent requirements and may facilitate recovery of the desired product in an advantageously high concentration.
In existing sample displacement chromatography separations, the sample components are introduced in the form of a homogeneous sample solution, so that the individual components are each delivered at a constant concentration throughout the sample application step. The driving force for separation is that weak binders are displaced from the limited number of binding sites on the stationary phase material by the more strongly binding bulk of the product. This proceeds in a continuous manner until the product and other stronger binders are fully retarded in the earlier part of the chromatography bed, thus permitting the more weakly binding impurities to stay bound to the stationary phase material further along the chromatography bed. Once all sample molecules are bound to the stationary phase, no further movement of these molecules will be observed.
A problem which may occur because of such use of homogeneous sample solutions, however, is that molecules of strongly binding components introduced during an early part of sample application may inadvertently be displaced by weaker binders introduced during a later stage of sample application. Since it is not possible for sample molecules to move against the carrier flow, such displaced strong binders may end up as impurities in the desired product part of the chromatography bed. Moreover, a weakly binding component entering the chromatography bed at a late stage of application may compete for a site at an early position of the bed; with only a small fraction of the total sample remaining, the likelihood of displacement from such an early position on the bed is decreased. Therefore this impurity may remain in an unexpectedly early position after the separation and so may contaminate the desired product. In a difficult separation, where several components have a close resemblance to the desired product, this effect may be substantial.
The present invention is based on the finding that loss of resolution in sample displacement chromatography separations resulting from this hitherto unrecognised problem may be significantly reduced if the sample components are applied to the chromatography bed in non-homogeneous form. In this way, a greater concentration of more weakly binding impurities may be introduced in the early part of sample application, with the early introduction of more strongly binding impurities being reduced; unwanted competition involving such impurities may therefore be substantially reduced, with a consequent improvement in product resolution.
Thus, according to one aspect of the present invention there is provided a method of sample displacement chromatography separation which comprises applying a multicomponent sample to one end of a chromatography bed comprising stationary phase material having affinity for components of the sample, causing components of the sample to become distributed along the chromatography bed by passage over the bed of a non-eluting mobile solvent phase, and recovering a desired component of the sample from at least a portion of the chromatography bed, characterised in that sample components are applied to the chromatography bed in a non-homogeneous manner such that the concentration of at least one component with relatively low affinity for the stationary phase material is enhanced during an earlier part of sample application and/or the concentration of at least one component with relatively high affinity for the stationary phase material is enhanced during a later part of sample application.
The desired non-homogeneous application of sample components may be achieved in a variety of ways, for example as described hereinafter; other methods may be devised by those skilled in the art armed with the disclosure of the present specification. In many instances non-homogeneity will be achieved using a preliminary chromatographic separation, either using sample displacement or elution chromatography. It should be emphasised that, whereas multiple chromatographic separations are well known in the art, existing procedures involve successive purifications of a partially purified fraction from a previous separation. This is entirely different from the procedure of the present invention, in which either the whole of a multicomponent sample or a substantial number of the components thereof is subjected to sample displacement chromatography in conjunction with some form of processing which induces an appropriate inhomogeneity in sample composition.
In existing chromatographic purifications involving either elution mode or sample displacement chromatography, it is common to discard fractions containing semi-pure product because of the presence of contaminating impurities which are difficult to remove at a later stage. In the process of the invention, on the other hand, such product-containing fractions from a first separation step may be utilised in subsequent sample displacement chromatography, since the impurity is present only in some of the fractions. It may therefore more efficiently be displaced if present in early-applied fractions or retained if present in late-applied fractions. It will be appreciated that such use of semi-pure product fractions may increase the overall yield as well as strengthening displacement effects.
The desired inhomogeneity in separation processes according to the invention may, for example, be achieved by using a solid sample and a carrier solvent which dissolves the sample components at different rates and thereby applies a concentration gradient to the chromatography bed. Since the solubility of sample components in the carrier solvents used in both normal and reversed phase chromatography will tend to decrease as the binding affinity of the components for the stationary phase material increases, the resulting solubility-induced concentration gradient will enhance the efficiency of the sample displacement separation in accordance with the invention.
Alternatively, where the sample components are already bound to a stationary phase as a result of a previous separation, the extraction conditions may be selected so that the principal component is extracted relatively late and therefore applied relatively late to the sample displacement chromatography bed. In this way the earlier extracted components are more efficiently displaced by this later-applied principal component.
In a further embodiment, a sample may be at least partially separated using a divisible sample displacement chromatography column system, for example as described in the aforementioned WO98/46623, with a number of successive fractions being collected by extraction from successive portions of stationary phase material. Representative systems which may be used here include reversed phase chromatography, extracting with an organic solvent system such as aqueous acetonitrile or acetic acid; ion exchange chromatography, extracting with a salt solution; or hydrophobic interaction chromatography, extracting by a decrease in salt concentration. Following any necessary removal of extracting materials, for example by removal of organic solvents or volatile salts or by operation of a desalting step, the fractions are then applied to the same or a similar chromatography system in order of increased binding affinity, i.e. with any eluted fractions and fractions from the outlet end of the initially used chromatography bed being applied first, and fractions from the inlet end of the initially used chromatography bed being applied last. Such delayed introduction of stronger displacers markedly enhances the efficiency of the second separation.
A still further embodiment may be used to counter the problem that slightly weaker binding impurities are often difficult to remove from the desired product because of tailing effects; this is particularly so when such an impurity constitutes a relatively large fraction of the sample, and arises because a significant amount of this impurity is applied to the chromatography bed at a late stage of the separation process. In such situations where a succession of separations is performed, the desired inhomogeneity may be achieved by applying one or more fractions from a previous separation or separations after application of the current sample to the chromatography bed. If the subsequently applied fraction or fractions are relatively enriched in the desired product, and possibly also in more strongly binding impurities, this will provide an additional separation force on the tailing impurity, resulting in an increased purity for the desired product. It will be appreciated that where a subsequently added fraction is itself in need of further purification, this will occur simultaneously with the above-described displacement of tailing impurity, thereby effectively increasing product throughput.
In a yet still further embodiment, the desired inhomogeneity may be generated by means of an elution mode separation. Thus, for example, gel permeation chromatography may be used to effect a first, partial separation; the eluate may then be applied either directly or in reverse order to the sample displacement chromatography bed, as appropriate. Alternatively, a not-fully resolving reversed phase chromatography separation using isocratic elution may be followed by sample displacement ion exchange chromatography; the proportion of organic solvent used in the first separation may advantageously be selected so that the desired product is principally eluted in a relatively early and concentrated fraction and is to some extent separated from impurities, e.g. with more hydrophobic impurities being retained on the reversed phase chromatography bed. If desired, the initial separation may also be used to remove certain unwanted sample components; thus, for example, salt impurities or partially protected synthetic products may be separated and discarded following reversed phase chromatography, in the latter case because protecting groups contribute to the hydrophobicity of a product. Fractions containing the desired product may then be subjected to sample displacement chromatography, e.g. using an ion exchange stationary phase material.
Representative areas in which separation procedures in accordance with the invention may be used include the following:
In many situations in modern drug design, enormous numbers of compounds are synthesised, often without the time or resources for proper purification or characterisation. A reliable and preferably automated purification system based on sample displacement chromatography may be used to handle several crude products individually and simultaneously, often with the benefit of avoiding other isolation steps.
If a crude peptide derived from solid phase synthesis is purified by cation exchange or reversed phase chromatography, an increased purity will be obtained. To ensure a consistently high purity product, the two steps may be combined in accordance with the process of the present invention. The content of failure sequences in the product may be reduced by the application of a mixture of target peptide having a free amino function in the N-terminal and shorter failure sequences that have been end capped by acetylation, a common procedure in peptide synthesis, in a system intended for sample displacement chromatography. The correct sequence will contain an extra positive charge at pH 2-3, when applied to a cation exchange chromatography bed with aqueous acetic acid as carrier. The capacity of the bed will govern the optimum loading of sample so that most of the product will remain bound to the bed and lower charged impurities will be displaced from the bed.
This first ion exchange chromatography purification may be followed by a reversed phase chromatography purification. The addition of salt to the carrier will extract the peptides from the ion exchange chromatography bed in order of increasing charge. Thus the remaining impurities will be transported onto the reversed phase chromatography bed ahead of the desired product, which will enter the bed relatively late, and so will displace the usually less hydrophobic impurities even more efficiently than when the sample is applied homogeneously.
The product from such a separation may be collected by extraction from the reversed phase chromatography bed, whereafter a high throughput, high resolution analytical system such as HPLC or capillary electrophoresis may be used to perform a purity check to enable combining of sufficiently pure product fractions.
Existing separation/purification procedures are frequently limited by insufficient selectivity which results in cumbersome elution mode separations having to be employed. Typical examples are when there is a small difference in apparent size in gel permeation chromatography or there are small differences in charge or hydrophobicity in isocratically eluted ion exchange and reversed phase chromatography systems respectively. Sample displacement chromatography may also be inefficient where the concentration, and thus the displacing effect, of the desired product in a given sample is low.
An example of this last situation is afforded by Veeraragavan (op. cit.), who endeavoured to purify soybean trypsin inhibitor by means of sample displacement anion exchange chromatography. From the published results, it is clear that the selectivity of the components was inadequate for an efficient sample displacement chromatography separation, so that elution mode gradient separation was employed as the main force for separation. Even so, the chemical purity of the product was not especially high, and recovery was insufficient for an efficient purification.
It can be seen from Veeraragavan""s gel electrophoresis results that the most strongly binding, i.e. late eluting, components (product peak 6 and impurity peak 8) appeared to have the lowest molecular weights and therefore the smallest sizes. An improved overall separation may therefore be achieved by running an initial gel permeation chromatography separation and feeding the eluate directly onto an anion exchange chromatography bed. The resulting late application of the xe2x80x9csize-sortedxe2x80x9d strongly binding components in the second separation step will significantly enhance the efficiency of this sample displacement chromatography step.
Gel permeation chromatography may be used in conjunction with a subsequent sample displacement step in the purification of a wide range of synthetic oligomers and polymers, where the principal impurities will differ in size from the desired product due to an insufficiency or excess of monomer residues, and will also differ in their binding affinities for site specific stationary phase material, for example as a result of differences in hydrophilicity/hydrophobicity, overall charge, charge density etc. It will be appreciated that the application order of eluted components from the gel permeation chromatography should be reversed in the event that late-eluting (i.e. smaller) component fractions from the gel permeation chromatography separation exhibit lower binding affinity for the stationary phase material in the sample displacement chromatography separation than do early eluting (i.e. larger) component fractions.
The above principle may be used in the purification of crude oligonucleotides, where full length sequences have a higher charge than shorter sequences and thereby bind more strongly in anion exchange chromatography, but where sample displacement chromatography alone may give insufficient resolution between a desired product and failure sequences such as an xe2x80x9cnxe2x88x921xe2x80x9d impurity. Similarly, in the purification of crude peptide samples by reversed phase sample displacement chromatography, initial gel permeation chromatography and flow reversal of the eluate therefrom may result in smaller less hydrophobic deletion sequences entering the reversed phase chromatography system somewhat ahead of the desired product; such impurities will therefore be much more efficiently displaced during sample displacement chromatography by the increased proportion of product in subsequently applied fractions. It will be appreciated that in such procedures the solvent used in the gel permeation chromatography step should be chosen so that it functions as the carrier in the subsequent sample displacement chromatography separation.
In an alternative procedure for the purification of synthetic oligonucleotides, reversed phase chromatography is used to induce the desired inhomogeneity. The retention time of the sample components on the reversed phase chromatography bed may be adjusted by addition of organic solvent or an ion pairing agent such as triethylamine buffer. Once the desired product begins to elute at basic pH, the eluate flow may be applied directly onto an anion exchange chromatography bed equilibrated with a suitable carrier. Following this sample displacement separation, the product may be extracted by addition of a salt solution and then passed through a desalting column. The desalting step, which in general will be based on gel filtration, may be designed to remove both low molecular weight salt and carrier solution from the previous separation, so as to give a simple aqueous solution of the desired product.
The purification of homopolymers may also be effected utilising the method of the present invention. Representative homopolymers include methoxy-polyethylene glycol (methoxy-PEG) compounds, whose size distribution may be modified by gel permeation chromatography combined with a reversed phase chromatography sample displacement separation. Such a procedure based on separation by size followed by size/hydrophobicity selection narrows the molecular weight range of the product. At the same time, bifunctional impurities which may cause cross-reactions in subsequent applications such as protein modification may also be removed.
Alternatively, PEG compounds may be purified by successive reversed phase chromatography sample displacement separations. A single reversed phase chromatography sample displacement step separates the components to a certain extent, but the selectivity may be insufficient to obtain a single oligomer in high purity. Using the procedure of the present invention, however, a sample may be separated into virtually monodisperse fractions. Thus, after a first separation, fractions of the original sample with narrowed size distribution may be collected from the various parts of the chromatography bed. Once reapplied in the order of migration (smallest molecule-containing fractions first), the displacement effect becomes more pronounced, with less risk of unwanted competition. Improved product purity is thereby obtained for each successive separation in a repetitive process. The whole sample may be reapplied, or parts of it may be removed, e.g. where a fraction contains a large portion of unwanted material or impurities. To increase efficiency, material from other fractionations may also be added, e.g. as hereinbefore described.
Such methods may be applied in industrial-scale processes to permit the economical preparation of highly pure polymer. It will be appreciated that both sample and solvents may be recovered after each separation cycle, thereby reducing operating costs.
In small scale gel permeation chromatography applications, any necessary flow reversal before a second separation step may simply be accomplished using a collection loop consisting of appropriate tubing and switching valves. After collection of eluate in the loop, a valve is switched and the collected sample is directed onto the second chromatography bed in reversed order. The actual volume to be reprocessed may be optimised in advance or may be determined by use of a detection system after the first chromatography bed. If larger volumes are to be processed, appropriate collection devices include fraction collection flasks connected with valves so that reapplication may be performed in reverse order of collection.
Large scale processes may advantageously be performed using a plurality of relatively short columns rather than one or a few longer columns. Thus, for example, it may be convenient to use a series of rectangular shaped columns having a 1xc3x972 m surface area and a bed height of 5-15 cm. Several such columns, for example 10 columns of 10 cm bed height, may be stacked on top of each other and connected with valves so as to be operable in series during sample application or individually during product extraction.
To ensure an even solvent distribution in columns with such a large cross-sectional area, it is desirable to position the inlet at one side on the top of the column and the outlet on the reverse side at the bottom of the column, the inlet and outlet each connecting with arrays of channels which facilitate even distribution of sample solution over the surfaces of the chromatography bed. All sample components will then be transported at an equal rate anywhere over the surface of the stationary phase material, and will pass through the bed by the shortest perpendicular route. Such large chromatography beds may be packed from above, and preferably incorporate spacer bars placed between opposite column walls and having the same height as the column bed height; these bars prevent the stationary phase material from moving sideways in the column and at the same time stabilise the top and bottom frits. Alternatively, for smaller scale separations and/or where stability of the chromatography bed is less critical, chromatography columns with side packing ports, e.g. as described in U.S. Pat. No. 5,667,676, may be employed.
A variety of devices may be constructed to facilitate interfacing with a following step. Thus, for example, a chromatography column device consisting of several columns drilled in a cylinder may be operated either in series or in parallel by rotation of top and bottom sealing plates having appropriate channels such that a sample solution may be passed from an inlet to the top of the first column, from the bottom of that column through a narrow bore tubing to the top of the following column, and similarly through all the columns. During sample extraction and column re-equilibration, the plates are rotated to align a common inlet and individual outlets with each column.
Products obtained from such a column arrangement may be collected in a device which facilitates their further processing. Such a device may be placed below the columns during fraction collection, with one position for each column. After fractions have been extracted and analysed, they may be processed further, for example by direct application to a new column in a suitable sequence. Alternatively, fractions may be concentrated on, for example, a Speedi-vac concentrator, or may be lyophilised in order to remove unwanted solvents.
To simplify material handling, the collecting device may include a hole in the bottom of each compartment to allow the sequential withdrawal of each sample fraction. The holes may be closed by a plate which prevents the fractions from leaking during previous handling; this plate may be rotatable so that one compartment at a time may be connected to a bottom outlet, e.g. fitted with appropriate connecting tubing. The application of pressure to the top of the device will assist in generating a flow of sample fraction from the connected compartment to, for example, a further chromatography bed. Such systems may be automated using simple valve switching, and may incorporate sensors which detect air-liquid interfaces and cause the plate to be turned one step to the next compartment once such an interface is detected.
Column devices may also be based on the 96-well microtiter plate format, the large number of fractions obtainable with this format being advantageous in applications of the process of the invention. If the system is automated, product fractions may be automatically re-injected in an appropriate sequence by a robotic system. The device may be constructed either as described in WO98/46623, or in a form in which a body of columns may be transported between different stations at which equilibration, separation or extraction may be performed by contacting different plates with the top and bottom of the body.
The column and collecting devices described above, with the exception of those described in WO98/46623 and U.S. Pat. No. 5,667,676, constitute a further feature of the present invention.
In general, a variety of stationary phase materials may be employed in the sample displacement chromatography step of separation procedures according to the invention. The requirement that the material has affinity for components of the sample is to be interpreted as requiring that the material has specific sites which are capable of reversible interaction with components of the sample. Gel filtration media, in which samples interact with networks rather than actual sites, are therefore inappropriate, but systems which may be used include straight and reversed phase chromatography, ion exchange chromatography, hydrophobic interaction chromatography and affinity chromatography.
Stationary phase materials used in separation processes according to the invention may be in any convenient form, for example as membranes, gels or microspheres, especially monodisperse microspheres, and will usually be packed into one or more columns, e.g. as hereinbefore described.
The requirement that the mobile solvent phase used in sample displacement separation processes in accordance with the invention is non-eluting is to be interpreted as indicating that the solvent is capable of transporting less-retarded sample components to the next available binding sites but has little or no ability to interfere with interactions between the dissolved sample components and the stationary phase material. A wide range of solvent systems may be used subject to this requirement. Aqueous systems useful in, for example, reversed phase and ion exchange chromatography include water, buffer solutions, solution of bases such as sodium hydroxide, ammonium bicarbonate or ammonium hydroxide, and solutions of acids such as acetic acid or trifluoroacetic acid.
It may be convenient to operate sample displacement separation steps in accordance with the invention at low pressure, for example as described in WO98/46623. Thus the mobile solvent phase may advantageously be applied at a pressure not exceeding 30 bar, e.g. less than 15 bar, preferably less than 10 bar. Operating pressures of around 3 bar may advantageously be employed for commercial separations; overpressures as low as 0.5 bar have been found to give good separations. It may be appropriate to use even lower or no overpressure, depending on the nature of the chromatography bed. Thus, for example, where the bed comprises relatively coarse material, e.g. a coarse polymer gel, the mobile solvent phase may be capable of movement as a result of gravity and/or interaction with the bed material; in such embodiments it may be appropriate to apply a very low overpressure such as 0.1 bar to control rather than to drive the flow of mobile solvent phase.
The pressure source which drives the mobile solvent phase may advantageously be a pressurised gas such as nitrogen, thereby avoiding the need for mechanical pumps and consequently reducing the operating costs of the process.
Recovery of the desired product following sample displacement separation may be achieved by any appropriate method, for example by extraction into an aqueous solvent system or a non-aqueous solvent system (e.g. an organic solvent or supercritical carbon dioxide), by use of a displacer solution having higher affinity for the stationary phase material than does the sample (e.g. aqueous acetonitrile or acetic acid in the case of reversed phase chromatography or a salt solution in the case of ion exchange chromatography), by a decrease in salt concentration (e.g. in the case of hydrophobic interaction chromatography), or by pH change. The use of steady state (i.e. non-gradient) conditions is preferred since it inherently permits the product to be obtained in more concentrated form than is possible using gradient elution. Moreover, since the recovery procedure is totally independent from the separation procedure, the conditions for recovery may be optimised to ensure maximum efficiency of product release and maximum efficiency of use of solvents, displacer solutions etc. without in any way compromising the efficiency of the separation. It is accordingly possible to recover products in concentrations 10- to 100-fold higher than those which may be obtained with gradient or isocratic elution.
The ability to extract products into solvents of choice is also advantageous in allowing recovery of products in stabilised form or in an optimum form for use in subsequent processing steps. Thus, for example, a protein may be extracted using buffer solution at a pH appropriate to maximise stability of the protein.


A biodegradable, biocompatible porous matrix as a scaffold for tissue engineering cartilage is formed of a copolymer of a polyalkylene glycol and an aromatic polyester such as a polyethylene glycol/polybutylene terephtalate copolymer. A ceramic coating such as a calcium phosphate coating may be provided on the scaffold by soaking the scaffold in a solution containing calcium and phosphate ions. A composite scaffold which is preferably a two-layer system may be formed having an outer surface of a layer of the porous matrix formed of the copolymer, and an outer surface of a layer of a ceramic material. The composite scaffold may be prepared by casting the copolymer on top of the ceramic material in a mould. Cells are preferably seeded on the scaffold prior to implanting, and the scaffold may contain bioactive agents that are released on degradation of the scaffold in vivo.

1. Field of the Invention
The invention relates to a scaffold for use in a method of tissue engineering cartilage.
2. Description of the Related Art
The inability of articular cartilage for self-repair is a major problem in the treatment of patients who have their joints damaged by traumatic injury or suffer from degenerative conditions, such as arthritis or osteoarthritis. Examples of currently employed treatments include subchondral drilling and abrasion. However, these treatments are hardly effective in the long term, as they do not promote formation of new or replacement cartilage tissue, or cartilage-like tissue. Instead, these treatments lead to scar or fibrous tissue, which cannot withstand joint loading in the long term. Thus, although the condition of patients treated by using these conventional techniques initially improves, eventually it will deteriorate, possibly leading to osteoarthritis.
Another therapy conventionally relied on for treating loss of cartilage is replacement with a prosthetic material, such as silicone for cosmetic repairs, or metal alloys for joint relinement. Placement of prostheses is commonly associated with significant loss of underlying tissue and bone without recovery of the full function allowed by the original cartilage, as well as the irritating presence of a foreign body. Other long term problems associated with a permanent foreign body can include infection, erosion and instability.
Recently, new approaches to cartilage tissue repair have been proposed. These approaches are based on implanting or injecting expanded autologous cells per se into a defect in a patient""s cartilage tissue. However, it has meanwhile been accepted that the majority of the thus implanted cells will not sustain. Also, this approach is only feasible for a relatively narrow group of patients.
Even more recent, it has been proposed in EP-A-0 469 070 to use a biocompatible synthetic polymeric matrix seeded with chondrocytes, fibroblasts or bone-precursor cells as an implant for cartilaginous structures. It is taught that it is essential that the polymeric matrix is formed of fibers or a fibrous mesh in order to provide free exchange of nutrients and waste products to the cells attached to the matrix. This free exchange is described to be particularly relevant in the stage after implantation wherein vascularization of the implant has not yet taken place. The material used for providing the polymeric matrix is a biocompatible synthetic material. The only specifically mentioned material is polyglactin 910, a 90:10 copolymer of glycolide and lactide.
The present invention aims to provide an improved scaffold for tissue engineering cartilage. It is an object to provide an artificial matrix which is highly suitable to serve as a temporary scaffold for cellular growth and implantation of cartilage. The matrix should be biodegradable and non-toxic and enable cell growth both in vivo and in vitro. It is a further object that the scaffold can provide sufficient mechanical strength for it to be utilized for cell growth to replace degenerated cartilage in joints, and desirably also to withstand joint loading. It should further be possible to design the scaffold such that it is suitable to replace hyaline or elastic cartilage in plastic and reconstructive surgery.
Surprisingly, it has been found that the above objectives are fulfilled by using a porous matrix of a specific polymeric material as a scaffold for engineering cartilage tissue. Thus, the invention relates to the use of a biodegradable, biocompatible porous matrix as a scaffold for tissue engineering cartilage, which matrix is formed of a copolymer of a polyalkylene glycol and an aromatic polyester.
The material used as a scaffold in accordance with the invention meets all the above requirements for use in cartilage repair or replacement. In particular, said material provides superior mechanical strength so that the scaffold is able to withstand joint loading to a degree which is not attainable using a fibrous structure.
Furthermore, the specific polymeric material on which the present scaffold is based has hydrogel properties and allows for diffusion through the material itself, in addition to diffusion through its porous structure. Of course, this feature is highly advantageous when cells are seeded onto the scaffold and are cultured thereon, as it enables a very efficient transport of nutrient and waste materials from and to the cells. Secondly, the material closely mimics the structure and properties of natural cartilage, which, containing 80% water, is also a hydrogel. Furthermore, the swelling behavior of the specific polymeric material allows for optimal fixation of the structure in a defect when it is implanted without cells seeded thereto in vitro,
A matrix to be used as a scaffold in accordance with the invention is biodegradable and biocompatible. In the context of the present invention, the term biocompatible is intended to refer to materials which may be incorporated into a human or animal body substantially without unacceptable responses of the human or animal. The term biodegradable refers to materials which, after a certain period of time, are broken down in a biological environment. Preferably, the rate of breakdown is chosen similar or identical to the rate at which the body generates autogenous tissue providing sufficient mechanical strength to replace the implant of which the biodegradable material is manufactured.
In accordance with the invention, the matrix has a slower rate of degradation in a biological environment than the copolymers of glycolide and lactide which are preferred according to the above discussed EP-A-0 469 070, ensuring mechanical support over the whole regeneration period in vivo before the extracellular matrix synthesized by cells seeded onto the scaffold, or by cells of the surrounding tissue present in vivo, takes over the mechanical function.
Further, the present matrix is porous (i.e. non-fibrous). This means that the matrix is a substantially homogeneous, solid structure, provided with small holes (pores), which enable diffusion of nutrients and waste products. As opposed to a fibrous structure, which is composed of different elements (fibers), the present porous matrix is a continuous structure, substantially composed of one element, comprising distinct compartments. It is preferred that the pores in the present matrix are interconnected.
Preferably, the matrix has a macroporosity between 30 and 99%, more preferably between 60 and 95%. The pores in the matrix preferably have a diameter of between 0.1 and 2000 xcexcm, more preferably between 1 and 1000 xcexcm. The macroporosity and the diameter of the pores will be chosen such that, on the one hand, sufficient diffusion of nutrients and waste products can take place, and, on the other hand, sufficient mechanical strength is provided by the matrix.
As has been mentioned, the present scaffold is formed of a specific class of polymeric materials having hydrogel properties. This is the class of copolymers of a polyalkylene glycol and an aromatic polyester. Preferably, these copolymers comprise 40-80 wt. %, more preferably 50-70 wt. % of the polyalkylene glycol, and 60-20 wt. %, more preferably 50-30 wt. % of the aromatic polyester. A preferred type of copolymers according to the invention is formed by the group of block copolymers.
Preferably, the polyalkylene glycol has a weight average molecular weight of from 150 to 4000, more preferably of 200 to 1500. The aromatic polyester preferably has a weight average molecular weight of from 200 to 5000, more preferably of from 250 to 4000. The weight average molecular weight of the copolymer preferably lies between 20,000 and 200,000, more preferably between 50,000 and 120,000. The weight average molecular weight may suitably be determined by gel permeation chromatography (GPC). This technique, which is known per se, may for instance be performed using tetrahydrofuran as a solvent and polystyrene as external standard.
In a preferred embodiment, the polyalkylene glycol component has units of the formula xe2x80x94OLOxe2x80x94COxe2x80x94Qxe2x80x94COxe2x80x94, wherein O represents oxygen, C represents carbon, L is a divalent organic radical remaining after removal of terminal hydroxyl groups from a poly(oxyalkylene)glycol, and Q is a divalent organic radical.
Preferred polyalkylene glycols are chosen from the group of polyethylene glycol, polypropylene glycol, and polybutylene glycol and copolymers thereof, such as poloxamers. A highly preferred polyalkylene glycol is polyethylene glycol.
The terms alkylene and polyalkylene generally refer to any isomeric structure, i.e. propylene comprises both 1,2-propylene and 1,3-propylene, butylene comprises 1,2-butylene, 1,3-butylene, 2,3-butylene, 1,2-isobutylene, 1,3-isobutylene and 1,4-isobutylene (tetramethylene) and similarly for higher alkylene homologues. The polyalkylene glycol component is preferably terminated with a dicarboxylic acid residue xe2x80x94COxe2x80x94Qxe2x80x94COxe2x80x94, if necessary to provide a coupling to the polyester component. Group Q may be an aromatic group having the same definition as R, or may be an aliphatic group such as ethylene, propylene, butylene and the like.
The polyester component preferably has units xe2x80x94Oxe2x80x94Exe2x80x94Oxe2x80x94COxe2x80x94Rxe2x80x94COxe2x80x94, wherein O represents oxygen, C represents carbon, E is a substituted or unsubstituted alkylene or oxydialkylene radical having from 2 to 8 carbon atoms, and R is a substituted or unsubstituted divalent aromatic radical.
In a preferred embodiment, the polyester is chosen from the group of polyethylene terephtalate, polypropylene terephtalate, and polybutylene terephtalate. A highly preferred polyester is polybutylene terephtalate.
It is believed that the use of a copolymer of polyethylene glycol, having a weight average molecular weight of between 800 and 1200 (preferably 1000) and polybutylene terephtalate in a weight ratio of between 65 to 35 and 75 to 25 (preferably 70 to 30) may lead to a faster proliferation of cells, such as chondrocytes, seeded on a scaffold of said copolymer.
The preparation of the copolymer will now be explained by way of example for a polyethylene glycol/polybutylene terephtalate copolymer. Based on this description, the skilled person will be able to prepare any desired copolymer within the above described class. An alternative manner for preparing polyalkylene glycol/polyester copolymers is disclosed in U.S. Pat. No. 3,908,201.
A polyethylene glycol/polybutylene terephtalate copolymer may be synthesized from a mixture of dimethyl terephtalate, butanediol (in excess), polyethylene glycol, an antioxidant and a catalyst. The mixture is placed in a reaction vessel and heated to about 180xc2x0 C., and methanol is distilled as transesterification proceeds. During the transesterification, the ester bond with methyl is replaced with an ester bond with butylene. In this step the polyethyene glycol substantially does not react. After transesterification, the temperature is raised slowly to about 245xc2x0 C., and a vacuum (finally less than 0.1 mbar) is achieved. The excess butanediol is distilled and a prepolymer of butanediol terephtalate condenses with the polyethylene glycol to form a polyethylene/polybutylene terephtalate copolymer. A terephtalate moiety connects the polyethylene glycol units to the polybutylene terephtalate units of the copolymer and thus such copolymer also is sometimes referred to as a polyethylene glycol terephtalate/polybutylene terephtalate copolymer (PEGT/PBT copolymer).
A porous structure of the polymeric material may be obtained by any known method, such as salt leaching or sintering. In principle, any combination of techniques, such as phase inversion, freeze drying and salt leaching may be used.
In a salt leaching procedure, the scaffold material may be subjected to a solvent casting procedure, wherein a substrate is formed by liquefying the material in a suitable solvent containing salt particles having a suitable particle size to acquire the desired pore size. The salt/polymer solution may then be cast on a plate using a substrate-casting apparatus fixed at a certain height finally leading to the desired thickness of the structure to be formed. The salt particles are then leached out of the copolymer by washing with, for instance (distilled) water.
It is also possible to use a viscous polymer gel in the salt leaching procedure, instead of a polymeric solution. In that case, the first step of the procedure is the preparation of a relatively concentrated polymer solution (preferably comprising at least 50 wt. % of the polymer) at elevated temperatures, such as from 60 to 120xc2x0 C. Preferred solvents have a relatively high boiling point, and are miscible with water. An example of a solvent which has been found to particular good results is N-methylpyrrolidone. In the second step of the procedure, salt particles are added to the polymer solution. Subsequently, the solution is transferred into a mould, having the desired shape and size of the scaffold to be formed, and is cooled to room temperature. Upon cooling, a stable polymer gel is formed. Placement of the gel in demineralized water leads to removal of the solvent and the salt, resulting in a stable porous material.
In a sintering procedure, the scaffold material may be placed into a mold and subsequently heated under pressure to a temperature below the melting point of the material. Releasing the pressure and cooling the material yields a sintered product. The skilled person may, based on his general knowledge, adapt the conditions during the sintering procedure such that the desired porous structure is obtained.
Yet another advantage of the specific polymeric material of which the present scaffold is formed is that it is possible to incorporate bioactive agents in said material, which agents are slowly released upon degradation of the material in vivo. In this regard, reference is made to the U.S. Pat. No. 5,980,948, the contents of which are incorporated herein by reference.
In a preferred embodiment, the outer surface of the scaffold is partly or completely provided with a ceramic coating. Preferably, the ceramic coating is a calcium phosphate coating. It has been found that the presence of a ceramic coating is highly beneficial to the attachment of cells to the scaffold. The calcium phosphate may be applied to the polymeric material by soaking said material into a highly concentrated calcifying solution at low temperature. The calcifying solution is preferably composed of at least is calcium and phosphate ions, and optionally of magnesium, carbonate, sodium and chloride ions, which are dissolved into water by bubbling carbon dioxide gas. During the natural release of carbon dioxide gas or its exchange with air, the pH of the calcifying solution is increased and the saturation is raised until the nucleation of carbonated calcium phosphate crystals on the surface of the scaffold. The process of bubbling/releasing CO2 gas through or from the calcifying solution can be repeated until a sufficient thickness of the coating has been reached. In general, the thickness of the ceramic layer will be between 0.1 and 20 xcexcm. It is preferred, that the ceramic coating is designed such that it has its beneficial effect during the seeding of cells onto the scaffold, and during the subsequent in vitro culturing of said cells. It is further preferred, that, by the time the scaffold is to be implanted into a patient""s body, the ceramic coating has substantially disappeared. This may for instance be accomplished through the presence of the cells or culture medium, e.g. by dissolution.
Under certain conditions, particularly when the scaffold is intended to be used in the treatment of full-thickness cartilage defects, comprising cartilage as well as bone, it has been found advantageous to use a composite scaffold comprising a first part which has an outer surface of a polymeric material, of the above discussed type, and second part which has an outer surface of a ceramic material. The composite matrix preferably is a two-layer system, wherein the ceramic part mimics the function of bone, and the polymeric part mimics the function of cartilage. Thus, the composite matrix mimics the nature of both cartilage and bone tissue. Furthermore, the ceramic outer surface facilitates adhesion of cells to the scaffold both in vitro and in vivo.
The first part of the composite matrix, is preferably substantially in its entirety formed of a copolymer of the above discussed type. The second part of the composite matrix may be substantially completely formed of a ceramic material. Examples of suitable ceramic materials include calcium phosphate, calcium carbonates, and sodium calcium phosphates. Particularly suitable ceramic materials are chosen from the group of octacalcium phosphate, apatites, such as hydroxyapatite and carbonate apatite, whitlockites, such as xcex1-tricalcium phosphate, xcex2-tricalcium phosphate, sodium calcium phosphate, and combinations thereof. It is also possible that the second part is formed of a different material, but coated with a ceramic material as set forth herein above. The said different material may be any type of polymeric material, preferably the above discussed copolymer of a polyalkylene glycol and an aromatic ester, or another suitable material, such as Bioglass or a glass-ceramic.
In a preferred embodiment, a dense layer, preferably of the above discussed copolymer of a polyalkylene glycol and an aromatic ester, is applied between the polymeric and ceramic parts to avoid movement of cells from one part to the other.
The composite may be prepared in any suitable manner, for instance by providing a mould having the desired shape of the composite scaffold. The ceramic part of the composite, which is prepared separately, may be placed in the mould, and the polymeric layer may be cast on top of the ceramic part, e.g. by injection moulding. It is also possible to apply the polymeric part in the form of a polymer-salt solution, which is used in the above described salt leaching procedure for achieving the desired porosity. Optionally, the dense layer may be applied onto the ceramic part first. A good attachment is obtained between the ceramic and polymeric parts of the composite as the polymeric material will invade the pores of the ceramic part to some extent during injection moulding.
A scaffold based on a biodegradable, biocompatible, porous matrix as described above, which is of course also encompassed by the present invention, may be used in tissue engineering cartilage with or without cells seeded thereon in vitro. The scaffold may be processed to have a particular desired form in any known manner. The matrix being formed of a polymeric material having hydrogel properties, the swelling behavior or the scaffold allows for swell fixation of the implant in a defect in cartilage tissue into which it is implanted. This swell fixation is specifically advantageous when the scaffold is implanted cell free. The extent of swelling can suitable be controlled by adjusting the composition of the polymeric material.
In a preferred embodiment, the scaffold is seeded with cells prior to its implantation. The cells may be any type of cells commonly occurring in natural cartilage or any type of cells capable of differentiating into cells commonly occurring in natural cartilage. Preferred cell types are chondrocytes, bone-precursor cells, stem cells, and cells of periosteum or perichondrium tissues. These cells may also be used in their crude form, e.g. in the form of bone marrow, comprising more than one cell type or even extracellular matrix. It is further preferred that the cells are autologous cells, thus minimizing, or even excluding, the chance of rejection responses in or disease transmission (e.g. HIV) to the patient treated with the present scaffold.
The seeding may be carried out in any known manner, for instance by static seeding. It is preferred, however, that the cells are seeded dynamically as has been described in co-pending European patent application 98203774.9, which is incorporated herein by reference. Subsequent to the seeding process, the cells are preferably cultured in vitro, allowing for a sufficient degree of proliferation and/or differentiation of the cells. The period required for culturing may vary broadly and range between one hour and several months, dependent on the number of seeded cells and the size of the implant required.
The invention further relates to the use of the above scaffold as a medical implant in cartilage repair. This use may specifically apply in cases of damaged cartilage in a patient as a result of inflammation, trauma, aging, or wherein the cartilage is congenitally defective.
The invention will now be elucidated by the following, non-restrictive example.

Scaffold for tissue engineering cartilage having outer surface layers of copolymer and ceramic material


A new and distinct Schlumbergera plant named ‘SAMBA BRAZIL’ particularly characterized by large upright to vertical flowers, with less reflexing of petals; flowers which have petals which are red-purple (RHS 58A) in color at the edges, color transitioning to orange (RHS 27D) and then white (RHS N155B) at the center of petal, and with a white (RHS N155C) throat; large quantity of flowers per plant; moderately vigorous growth rate and fairly compact, freely branching growth habit; and ovoid to lanceolatoid buds red-purple (RHS N66A) in color.

The present invention relates to a new and distinct cultivar of Schlumbergera plant, botanically known as Schlumbergera truncata (Haworth) Moran., commonly known as Thanksgiving Cactus, and hereinafter referred to by the cultivar name 'SAMBA BRAZIL'.
Schlumbergera (formerly Zygocactus) of the Cactaceae family consists of 6 known species which are epiphytic cacti and native to Brazil. Common names for Schlumbergera plants include: Crab Cactus for the cultivar's claw-like phylloclade margin, Thanksgiving Cactus for cultivars which bloom in November, and Christmas Cactus for cultivars which bloom in December.
The new Schlumbergera cultivar is a product of a planned breeding program conducted by the inventor, Lau Lindegaard RASMUSSEN, in Fyn, Demark. The objective of the breeding program was to develop a new Schlumbergera cultivar with excellent branching habit and large, upright flowers with a unique color combination.
The new Schlumbergera cultivar originated from an outcrossing made by the. inventor, Lau Lindegaard RASMUSSEN, in 2001 in Fyn, Demark. The female or seed parent is the Schlumbergera truncata 'SALSA DANCER' (unpatented, disclosed in EU-CPVO No. 2004/1247). The male or pollen parent is an Schlumbergera truncata '8620A' (unpatented). The new Schlumbergera cultivar was discovered and selected by the inventor as a single flowering plant within the progeny of the stated outcrossing in a controlled environment in 2003 in Fyn, Denmark, on the basis of its flower color and its fairly compact and freely branching habit. Plants of the new Schlumbergera are more upright, and have a unique color combination of the flowers combined with healthy, shiny green phyllocladia and excellent branching.
Asexual reproduction of the new Schlumbergera cultivar by phylloclade cuttings, followed by trial production batches, was first performed in January of 2004 in Fyn, Denmark, and has demonstrated that the combination of characteristics as herein disclosed for the new cultivar are firmly fixed and retained through successive generations of asexual reproduction. The new cultivar reproduces true to type.


The disclosed embodiments include an aquarium having a low-current filtration system. In one aspect, an apparatus includes a tank having a closed end and an open end, a base that receives and supports the closed end, and a lid removably positioned over the open end. The apparatus also includes a filter base disposed onto an interior surface of the closed end, and a filter housing that engages a housing support structure disposed onto the filter base. The filter housing includes a filter cartridge separating a first chamber from a second chamber, a pump disposed within the second chamber, and a reservoir disposed within the second chamber at a distal end of the filter housing. The reservoir includes an outlet structure having multiple discharge locations, and is connected to the pump across an elongated tube.

Aquariums housing aquatic life provide an aesthetically pleasing, relaxing display of motion and color that promote a viewer's curiosity and interest. Certain aquatic life, including species of fish commonly housed in aquariums, thrive in aquatic environments characterized by minimal hydrodynamic current and fluid motion. Nonetheless, aquarium maintenance, including proper water filtration and circulation, is essential to establishing a healthy aquatic environment for these fish species. Many conventional aquarium filtration systems face challenges in balancing the demands of proper water filtration and circulation with the desired fluid-dynamic characteristics of the aquatic environment.

Aquarium and non-disruptive filtration system